The Tpl-2 locus. cloned by provirus tagging from a subline of the MoMuLV induced rat thymoma 2769, defines a gene encoding a protein kinase which is expressed primarily in spleen, thymus and lung. Tpl-2 spans a 35 kb genomic DNA region and contains eight exons. During tumor progression provirus insertion occurs in the last intron and in the same transcriptional orientation with the Tpl-2 gene in 22.5% of the thymomas. This leads to the enhanced expression of a truncated RNA transcript in which the 3' exon is replaced by intron and proviral LTR sequences. This transcript encodes a protein in which the 43 C-terminal amino acids encoded by the eighth exon are replaced by seven amino acids encoded by the last intron. This genetic change confers growth advantage to affected cell clones. Cultivation of three sublines derived from tumor 2769, selected cells which harbor independent provirus insertions in the Tpl-2 locus. One of these cell lines, upon continuing cultivation, underwent further selection for cells expressing a short (2 kb) Tpl-2 mRNA transcript, suggesting that the short RNA may encode a protein with enhanced oncogenic activity. Given that rearrangements of the Tpl-2 locus were frequently detected in primary tumors, we suggest that the selection of cells with such rearrangements operates not only in culture but also in vivo. Exposure of normal rat spleen cells to concanavalin A (ConA) induces the expression of Tpl-2 within the first 60 minutes from the time of exposure. This finding, which was observed both in the presence and absence of cycloheximide, suggests that, in normal splenocytes, Tpl-2 may be involved in the transition from a quiescent to the G1 phase of the cell cycle. Tpl-2 is unique in that although it is a serine/threonine kinase, it harbors an N-terminal domain exhibiting distant homology to the SH2 region of cytoplasmic tyrosine kinases. In vitro binding assays revealed that the Tpl-2 SH2-like domain binds Src but falls to bind a kinase(-) mutant of Src suggesting that binding may be phosphotyrosine dependent. Proteins harboring the Tpl-2 type SH2-like domain are encoded by multiple genes in all species from insects to mammals. A protein fragment that extends between amino acids 1 and 290 was expressed in E. coli as a fusion with the Maltose binding protein (MBP). After cleavage with activated coagulation factor X, the Tpl-2 portion of the fusion protein was used to raise polyclonal antibodies in rabbits. The antibodies detect a 55 kd protein exhibiting kinase activity. Tpl-2 was mapped near the centromere on rat chromosome 17, mouse chromosome 18 and human chromosome l0p11. The Tpl-2 map location in humans defines a region characterized by karyotypic abnormalities associated with adult T cell leukemia (ATL) and acute non- lymphocytic leukemia (ANLL). The same region could also be involved in the hereditary syndrome, multiple endocrine neoplasia type II.